Pro-Survival Pathway Protects from C. difficile Toxin-Mediated Cell Death.

There is an urgent need for new non-antibiotic based treatment strategies for Clostridioides difficile infection. C. difficile toxin B (TcdB) is a virulent factor that is essential for causing disease. Here, we investigated whether a survival-signaling pathway could protect against TcdB. We found significant increase in caspase-3 apoptotic activity in intestinal epithelial cells of mice exposed to TcdB. Subsequently, activation of the MIF-CD74-Akt pro-survival signaling pathway blocked TcdB-induced caspase-3 activity and intestinal epithelial cell death. This brief report provides proof-of-concept that targeting pro-survival pathways may represent a unique antibiotic-independent strategy for protecting against C. difficile toxin-mediated cell death.

Infection and inflammation stimulate expansion of a CD74+ Paneth cell subset to regulate disease progression.

Paneth cells (PCs), a specialized secretory cell type in the small intestine, are increasingly recognized as having an essential role in host responses to microbiome and environmental stresses. Whether and how commensal and pathogenic microbes modify PC composition to modulate inflammation remain unclear. Using newly developed PC-reporter mice under conventional and gnotobiotic conditions, we determined PC transcriptomic heterogeneity in response to commensal and invasive microbes at single cell level. Infection expands the pool of CD74+ PCs, whose number correlates with auto or allogeneic inflammatory disease progressions in mice. Similar correlation was found in human inflammatory disease tissues. Infection-stimulated cytokines increase production of reactive oxygen species (ROS) and expression of a PC-specific mucosal pentraxin (Mptx2) in activated PCs. A PC-specific ablation of MyD88 reduced CD74+ PC population, thus ameliorating pathogen-induced systemic disease. A similar phenotype was also observed in mice lacking Mptx2. Thus, infection stimulates expansion of a PC subset that influences disease progression.

Clostridioides difficile Infection in Children: Recent Updates on Epidemiology, Diagnosis, Therapy.

Clostridioides (formerly Clostridium) difficile is the most important infectious cause of antibiotic-associated diarrhea worldwide and a leading cause of healthcare-associated infection in the United States. The incidence of C. difficile infection (CDI) in children has increased, with 20 000 cases now reported annually, also posing indirect educational and economic consequences. In contrast to infection in adults, CDI in children is more commonly community-associated, accounting for three-quarters of all cases. A wide spectrum of disease severity ranging from asymptomatic carriage to severe diarrhea can occur, varying by age. Fulminant disease, although rare in children, is associated with high morbidity and even fatality. Diagnosis of CDI can be challenging as currently available tests detect either the presence of organism or disease-causing toxin but cannot distinguish colonization from infection. Since colonization can be high in specific pediatric groups, such as infants and young children, biomarkers to aid in accurate diagnosis are urgently needed. Similar to disease in adults, recurrence of CDI in children is common, affecting 20% to 30% of incident cases. Metronidazole has long been considered the mainstay therapy for CDI in children. However, new evidence supports the safety and efficacy of oral vancomycin and fidaxomicin as additional treatment options, whereas fecal microbiota transplantation is gaining popularity for recurrent infection. Recent advancements in our understanding of emerging epidemiologic trends and management of CDI unique to children are highlighted in this review. Despite encouraging therapeutic advancements, there remains a pressing need to optimize CDI therapy in children, particularly as it pertains to severe and recurrent disease.

Alcohol Abuse Drug Disulfiram Is Effective against Cyst Stages of Entamoeba histolytica Parasite.

New anti-Entamoeba histolytica multistage drugs are needed because only one drug class, nitroimidazoles, is available for treating invasive disease, and it does not effectively eradicate the infective cyst stage. Zinc ditiocarb (ZnDTC), a main metabolite of the FDA-approved drug disulfiram, was recently shown to be highly effective against the invasive trophozoite stage. In this brief report, we show that ZnDTC is active against cysts, with similar potency to first-line cysticidal drug paromomycin.

Drug Repurposing of the Alcohol Abuse Medication Disulfiram as an Anti-Parasitic Agent.

Parasitic infections contribute significantly to worldwide morbidity and mortality. Antibiotic treatment is essential for managing patients infected with these parasites since control is otherwise challenging and there are no vaccines available for prevention. However, new antimicrobial therapies are urgently needed as significant problems exist with current treatments such as drug resistance, limited options, poor efficacy, as well as toxicity. This situation is made worse by the challenges of drug discovery and development which is costly especially for non-profitable infectious diseases, time-consuming, and risky with a high failure rate. Drug repurposing which involves finding new use for existing drugs may help to more rapidly identify therapeutic candidates while drastically cutting costs of drug research and development. In this perspective article, we discuss the importance of drug repurposing, review disulfiram pharmacology, and highlight emerging data that supports repurposing disulfiram as an anti-parasitic, exemplified by the major diarrhea-causing parasite Entamoeba histolytica.

COVID-19 and Corticosteroids: Unfamiliar but Potentially Fatal Infections That Can Arise following Short-Course Steroid Treatment.

Corticosteroid use is increasing worldwide as recent studies confer survival benefit of corticosteroids in the management of patients with severe COVID-19. Strongyloides and amebic infections are neglected diseases that can progress to catastrophic complications in patients exposed to corticosteroids, even with short treatment courses. To prevent lethal outcomes, clinicians should be aware of the threat these two parasitic infections pose to at-risk patients receiving corticosteroids, especially in the era of COVID-19.

Acute appendicitis in four children with SARS-CoV-2 infection.

We describe 4 children (11-17 years in age) at our institution with acute appendicitis in the setting of SARS-CoV-2 infection, suggesting a possible association. Providers should consider testing for this infection in patients with severe gastrointestinal symptoms, in order to take appropriate transmission based precautions, until more is understood.

COP9 signalosome is an essential and druggable parasite target that regulates protein degradation.

Understanding how the protozoan protein degradation pathway is regulated could uncover new parasite biology for drug discovery. We found the COP9 signalosome (CSN) conserved in multiple pathogens such as Leishmania, Trypanosoma, Toxoplasma, and used the severe diarrhea-causing Entamoeba histolytica to study its function in medically significant protozoa. We show that CSN is an essential upstream regulator of parasite protein degradation. Genetic disruption of E. histolytica CSN by two distinct approaches inhibited cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin in a neddylated state, disrupting UPS activity and protein degradation. In addition, zinc ditiocarb (ZnDTC), a main metabolite of the inexpensive FDA-approved globally-available drug disulfiram, was active against parasites acting in a COP9-dependent manner. ZnDTC, given as disulfiram-zinc, had oral efficacy in clearing parasites in vivo. Our findings provide insights into the regulation of parasite protein degradation, and supports the significant therapeutic potential of COP9 inhibition.

Role of MIF Cytokine/CD74 Receptor Pathway in Protecting Against Injury and Promoting Repair.

Wound healing after an injury is essential for life. An in-depth understanding of the healing process is necessary to ultimately improve the currently limited treatment options for patients suffering as a result of damage to various organs and tissues. Injuries, even the most minor, trigger an inflammatory response that protects the host and activates repair pathways. In recent years, substantial progress has been made in delineating the mechanisms by which inflammatory cytokines and their receptors facilitate tissue repair and regeneration. This mini review focuses on emerging literature on the role of the cytokine macrophage migration inhibitory factor (MIF) and its cell membrane receptor CD74, in protecting against injury and promoting healing in different parts of the body.

The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function.

Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab1's deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.

CD74 Signaling Links Inflammation to Intestinal Epithelial Cell Regeneration and Promotes Mucosal Healing.

BACKGROUND & AIMS: The inflammatory response to intestinal damage promotes healing through mechanisms that are incompletely understood. Gene expression of cluster of differentiation 74 (CD74), the receptor for cytokine macrophage migration inhibitory factor, is increased in patients with inflammatory bowel disease (IBD), however, the role of CD74 signaling in intestinal inflammation remains poorly understood. The aim of this study was to determine the functional role of CD74 signaling in intestinal inflammation. METHODS: We studied the characteristics of CD74 protein expression in human IBD and experimental colitis. The functional role of CD74 signaling in the intestine was investigated using cellular models; wild-type, CD74-/-, and bone marrow chimera mice; neutralizing anti-CD74 antibodies; flow cytometry; immunohistochemistry; immunofluorescence; immunoblotting; and clustered regularly interspaced short palindromic repeats and associated protein 9 technology. RESULTS: In IBD patients and experimental colitis, CD74-receptor protein expression was increased in inflamed intestinal tissue, prominently in the crypt epithelial cells. By using distinct but complementary chemical and non-chemically induced mouse models of colitis with genetic and antibody neutralization approaches, we found that CD74 signaling was necessary for gut repair. Mechanistically, we found that the macrophage migration inhibitory factor cytokine, which also is increased in colitis, stimulated the CD74 receptor, enhancing intestinal epithelial cell proliferation through activation of the protein kinase B and the extracellular signal-regulated kinase pathways. Our data also suggest that CD74 signaling in immune cells was not essential for mucosal healing. CONCLUSIONS: CD74 signaling is strongly activated during intestinal inflammation and protects the host by promoting epithelial cell regeneration, healing, and maintaining mucosal barrier integrity. Enhancing the CD74 pathway may represent a unique therapeutic strategy for promoting healing in IBD.

Purification of Antibodies Against Entamoeba histolytica MIF and Their Use in Analyzing Human and Mouse Samples.

Macrophage migration inhibitory factor (MIF) is a proinflammatory and proproliferative cytokine expressed in humans. MIF homologs also exist in many pathogenic protozoans, including Entamoeba, Plasmodium, Toxoplasma, and Leishmania. Production of antibodies against parasite proteins allows for the generation of assays to measure and visualize parasite infection within hosts. In this chapter, we describe how to specifically purify antibodies against Entamoeba histolytica MIF (EhMIF), and subsequently use anti-EhMIF antibodies for ELISA on mouse and human samples and for immunohistochemistry on human tissue. These methods can be applied to any protein for high-quality antibody purification.

Targeting Parasite-Produced Macrophage Migration Inhibitory Factor as an Antivirulence Strategy With Antibiotic-Antibody Combination to Reduce Tissue Damage.

Targeting virulence factors represents a promising alternative approach to antimicrobial therapy, through the inhibition of pathogenic pathways that result in host tissue damage. Yet, virulence inhibition remains an understudied area in parasitology. Several medically important protozoan parasites such as Plasmodium, Entamoeba, Toxoplasma, and Leishmania secrete an inflammatory macrophage migration inhibitory factor (MIF) cytokine homolog, a virulence factor linked to severe disease. The aim of this study was to investigate the effectiveness of targeting parasite-produced MIF as combination therapy with standard antibiotics to reduce disease severity. Here, we used Entamoeba histolytica as the model MIF-secreting protozoan, and a mouse model that mirrors severe human infection. We found that intestinal inflammation and tissue damage were significantly reduced in mice treated with metronidazole when combined with anti-E. histolytica MIF antibodies, compared to metronidazole alone. Thus, this preclinical study provides proof-of-concept that combining antiparasite MIF-blocking antibodies with current standard-of-care antibiotics might improve outcomes in severe protozoan infections.

Case Report: Lower Gastrointestinal Bleeding due to Entamoeba histolytica Detected Early by Multiplex PCR: Case Report and Review of the Laboratory Diagnosis of Amebiasis.

We report a case of Entamoeba histolytica infection in a young man who presented with cerebral infarction and shortly after admission developed bloody diarrhea with fever. A rapid diagnosis of severe E. histolytica colitis was established through the use of a multiplex polymerase chain reaction enteropathogen stool panel. This result was unexpected in a patient native to the United States without known risk factors for amebiasis and negative stool microscopy examination for ova and parasites. Rapid diagnosis allowed prompt initiation of appropriate anti-amebic therapy and ultimately a good outcome in a condition that otherwise carries high morbidity and fatality.

Parasite-Produced MIF Cytokine: Role in Immune Evasion, Invasion, and Pathogenesis.

Protozoan parasites represent a major threat to health and contribute significantly to morbidity and mortality worldwide, especially in developing countries. This is further compounded by lack of effective vaccines, drug resistance and toxicity associated with current therapies. Multiple protozoans, including Plasmodium, Entamoeba, Toxoplasma, and Leishmania produce homologs of the cytokine MIF. These parasite MIF homologs are capable of altering the host immune response during infection, and play a role in immune evasion, invasion and pathogenesis. This minireview outlines well-established and emerging literature on the role of parasite MIF homologs in disease, and their potential as targets for therapeutic and preventive interventions.

Tissue Destruction Caused by Entamoeba histolytica Parasite: Cell Death, Inflammation, Invasion, and the Gut Microbiome.

PURPOSE OF REVIEW: Entamoeba histolytica is a protozoan parasite that causes amebiasis, which remains a significant cause of morbidity and mortality worldwide. E. histolytica causes tissue destruction which leads to clinical disease. This review outlines some of the recent advances that have furthered our understanding of the processes that lead to the tissue damage caused by E. histolytica. RECENT FINDINGS: Recent studies have identified new mechanisms involved in E. histolytica-induced tissue damage. These include (i) new form of contact-dependent killing called trogocytosis; (ii) parasite-produced cytokine, macrophage migration inhibitory factor, that contributes to inflammation; (iii) exploitation of host immune response to promote invasion; and (iv) the contribution of the gut microbiome to clinical disease. SUMMARY: Targeting these mechanisms that result in tissue injury should be a focus of future research for the development of improved preventive and therapeutic strategies for amebiasis.

The Impact of Systemic Inflammation on Neurodevelopment.

Inflammatory mediators affect the brain during development. Neurodevelopmental disorders such as autism spectrum disorders, cognitive impairment, cerebral palsy, epilepsy, and schizophrenia have been linked to early life inflammation. Recent advances have shown the effects of systemic inflammation on children's neurodevelopment. We discuss the potential mechanisms by which inflammatory molecules can exert their effects on the developing brain and consider the roles of MHC class I molecules, the HPA axis, glial cells, and monoamine metabolism. Methods to prevent the effects of cytokine imbalance may lead to the development of new therapeutics for neuropsychiatric disorders. Future research should focus on identifying at-risk individuals and early effective interventions to prevent long-term neurodevelopmental disabilities.

Interaction between parasite-encoded JAB1/CSN5 and macrophage migration inhibitory factor proteins attenuates its proinflammatory function.

Multiple protozoans produce homologs of the cytokine MIF which play a role in immune evasion, invasion and pathogenesis. However, how parasite-encoded MIF activity is controlled remains poorly understood. Cytokine activity can be inhibited by intracellular binding partners that are released in the extracellular space during cell death. We investigated the presence of an endogenous parasite protein that was capable of interacting and interfering with MIF activity. A screen for protein-protein interaction was performed using immunoaffinity purification of amebic cell lysate with specific anti-Entamoeba histolytica MIF (EhMIF) antibody followed by mass spectrometry analysis, which revealed an E. histolytica-produced JAB1 protein (EhJAB1) as a potential binding partner. JAB1 was found to be highly conserved in protozoans. Direct interaction between the EhMIF and EhJAB1 was confirmed by several independent approaches with GST pull-down, co-immunoprecipitation, and Biolayer interferometry (BLI) assays. Furthermore, the C-terminal region outside the functional JAMM deneddylase motif was required for EhMIF binding, which was consistent with the top in silico predictions. In addition, EhJAB1 binding blocked EhMIF-induced IL-8 production by human epithelial cells. We report the initial characterization of a parasite-encoded JAB1 and uncover a new binding partner for a protozoan-produced MIF protein, acting as a possible negative regulator of EhMIF.

A Review of the Global Burden, New Diagnostics, and Current Therapeutics for Amebiasis.

Amebiasis, due to the pathogenic parasite Entamoeba histolytica, is a leading cause of diarrhea globally. Largely an infection of impoverished communities in developing countries, amebiasis has emerged as an important infection among returning travelers, immigrants, and men who have sex with men residing in developed countries. Severe cases can be associated with high case fatality. Polymerase chain reaction-based diagnosis is increasingly available but remains underutilized. Nitroimidazoles are currently recommended for treatment, but new drug development to treat parasitic agents is a high priority. Amebiasis should be considered before corticosteroid therapy to decrease complications. There is no effective vaccine, so prevention focuses on sanitation and access to clean water. Further understanding of parasite biology and pathogenesis will advance future targeted therapeutic and preventative strategies.